A new luminescent probe enables sensitive and specific detection of changes in Ca2+ concentrations in cells.

Optogenetics is term applied to a group of techniques that enables one to use light to control cellular processes. Most often, it involves the expression of a light-sensitive protein, such as an ion channel, in a target cell and then using light to stimulate the activity of that protein. Thus far, the major application has been to study ion fluxes and the effects of those fluxes in neurons. The approach provides a highly effective way to modulate cellular ion fluxes, but a method to measure those fluxes is also required. In the past, the typical approach to achieve this goal has been through the use of fluorescent sensors that respond to the ion of interest, most often Ca2+. However, the use of these sensors in optogenetics studies poses some challenges. In particular the light required to activate the fluorescent sensor may aberrantly stimulate the optogenetic sensor as well, triggering unwanted responses. To solve this problem, Vanderbilt Institute of Chemical Biology member Carl Johnson and his laboratory have developed a new luminescent probe to monitor ion fluxes in optogenetics experiments [J. Yang, et al. (2016) Nat. Commun., 7, 13268].

The primary advantage of a luminescent sensor over a fluorescent sensor is that the luminescent sensor does not require light in order to generate a signal. Instead, it produces light via an enzymatic chemical reaction. Thus, the potential for unintentionally stimulating the optogenetics sensor is minimized. Ideally, a luminescent sensor would be highly sensitive, specific for the ion to be monitored, genetically encoded, and ratiometric. The term ratiometric means that the response is based on the ratio of the light emitted at two different wavelengths rather than the absolute intensity of the light at a single wavelength. Ratiometric sensors have the advantage that they provide the same response for a given ion concentration regardless of the concentration of the sensor. To achieve a Ca2+ sensor exhibiting these characteristics, the investigators genetically encoded a protein (CalFluxVTN) comprising the NanoLuc luciferase and the Venus fluorescent protein separated by the troponin-C calcium sensor linked by short peptide sequences (Figure 1a). The expressed CalFluxVTN protein is capable of undergoing a Ca2+-dependent conformational change in its troponin-C region that brings the NanoLuc luciferase and the Venus fluorescent protein closer together. This enables a transfer of NanoLuc-generated luminescent energy to Venus, which then emits light at a lower energy wavelength through a process known as bioluminescence resonance energy transfer (BRET). BRET results in a Ca2+-mediated reduction in luminescence at the NanoLuc wavelength, and an increase in luminescence at the Venus wavelength so that the ratio of luminescence at the two wavelengths is directly related to the Ca2+ concentration (Figure 1). The investigators evaluated multiple permutations of possible NanoLuc/Venus/troponin-C configurations to optimize the CalFluxVTN structure for brightness and dynamic BRET range. In vitro studies revealed that CalFluxVTN is 30-50-fold brighter than the best available luminescent Ca2+ sensor (Nano-lantern(Ca2+)), and that its 11-fold change in BRET ratio over a Ca2+ concentration of 0 to 39 μM is far superior to the 1.3-fold change in intensity exhibited by Nano-lantern(Ca2+). In addition, CaFluxVTN does not respond to Mg2+, H+, K+, or Na+. Thus, it appears that CalFluxVTN exhibits all of the desired qualities of a luminescent Ca2+ sensor.

To test CalFluxVTN's ability to sense Ca2+ fluxes in cells, the investigators first expressed the protein in HeLa cells. They were able to confirm the presence of intact Venus through its characteristic fluorescence and NanoLuc through its luminescence upon addition of its substrate furimazine. The cells also emitted light of the correct wavelength for BRET between the two portions of CalFluxVTN (Figure 2). Even more important, when the cells were exposed to histamine, a stimulus known to evoke Ca2+ fluxes, appropriate changes in BRET signals ensued (Figure 2).

FIGURE 2. Micrographs of HeLa cells expressing CalFluxVTN. "BF" is a bright-field image of the cells. "Venus Fluor" confirms the fluorescence of the Venus portion of the protein, shown in pseudo-color green. "Nluc Lumi" confirms the luminescence of the NanoLuc portion of the protein upon addition of the substrate furimazine. "BRET" shows the luminescence of the protein at 525 nm due to the transfer of energy between NanoLuc and Venus. The two images on the right show ratiometric data for BRET before (t = 86 s) and after (t = 106 s) the addition of histamine. These images are color-coded to indicate the observed ratios with the key shown on the right. Figure reproduced under a Creative Commons Attribution 4.0 International License from J. Yang, et al. (2016) Nat. Commun., 7, 13268.

In their next set of experiments, the investigators evaluated the effectiveness of their new sensor in conjunction with optogenetics using HEK293 cells. They expressed both CalFluxVTN and melanopsin, a light-sensitive protein that induces Ca2+ fluxes in cells upon exposure to blue light. They observed a strong BRET signal in the cells upon blue light exposure (Figure 3), and a series of control experiments confirmed that this signal was, indeed, a response to light-dependent Ca2+ fluxes generated by melanopsin. When they performed the same experiments using the fluorescent sensor GCaMP6s instead of CalFluxVTN, they were also able to detect a light-dependent signal. However, the response of GCaMP6s was much more varied, resulting in a larger error than that observed with CalFluxVTN. The investigators hypothesized that the poorer response of GCaMP6s was due to the requirement to constantly irradiate the cells with light in order to observe the fluorescent signal, potentially resulting in aberrant stimulation of melanopsin.

As most optogenetics experiments are performed using neurons or neuronal tissue, the researchers next expressed CalFluxVTN in primary rat hippocampal neurons. They successfully demonstrated a Ca2+-dependent BRET response in these cells upon exposure to a depolarizing high K+environment. This promising result led them to express CalFluxVTN in the hippocampi of live mice through direct injection of a adenovirus-associated vector (AAV) carrying the gene for the sensor into the brain. Hippocampal slices isolated from the mice exhibited both Venus-dependent fluorescence and NanoLuc-dependent luminescence. They also exhibited a reversible BRET signal in response to Ca2+ fluxes triggered by exposure to high K+ concentrations in the medium (Figure 4).

FIGURE 4. Photomicrographs of of hippocampal slices obtained from mice pretreated with an AAV vector encoding CalfluxVTN. "Venus Fluor" confirms the fluorescence of the Venus portion of the protein. "Nluc Lumi" confirms the luminescence of the NanoLuc portion of the protein, and "BRET" confirms the transfer of energy between NanoLuc and Venus. Micrographs labeled t = 30.05 s and t = 34.25 s show the ratiometric response of the tissue (key to the right) for slices before and after exposure to high K+, respectively. Figure reproduced under a Creative Commons Attribution 4.0 International License from J. Yang, et al. (2016) Nat. Commun., 7, 13268.

To further confirm the potential usefulness of CalFluxVTN in optogenetics studies, the investigators expressed the sensor along with CheRiff, a variant of channelrhodopsin-2 in primary rat hippocampal neurons. The CheRiff protein is a light-sensitive ion pore that conducts multiple positive ions, including Ca2+ into cells. The transfected cells exhibited a strong BRET response to blue light pulses (Figure 5). Notable findings were the brightness of the signal that enabled faster recording rates and higher temporal resolution than could be obtained with more conventional fluorescent ion sensors.

The results support the conclusion that CalFluxVTN meets all of the criteria for a luminescent Ca2+sensor that can be used in conjunction with optogenetics approaches. Furthermore, it's potential applications are not limited to optogenetics experiments. We expect that the availability of this sensor will facilitate many future studies of ion fluxes in neurons as well as a range of other tissues and cells.